Pages 615-631Review
Kai Kaarniranta, Antero Salminen, Annakaisa Haapasalo, Hilkka Soininen, Mikko HiltunenAge-Related Macular Degeneration (AMD): Alzheimer’s Disease in the Eye?Abstract: Age-related macular degeneration (AMD) is a late-onset, neurodegenerative retinal disease that shares several clinical and pathological features with Alzheimer's disease (AD), including stress stimuli such as oxidative stress and inflammation. In both diseases, the detrimental intra- and extracellular deposits have many similarities. Aging, hypercholesterolaemia, hypertension, obesity, arteriosclerosis, and smoking are risk factors to develop AMD and AD. Cellular aging processes have similar organelle and signaling association in the retina and brain tissues. However, it seems that these diseases have a different genetic background. In this review, differences and similarities of AMD and AD are thoroughly discussed.

Pages 633-642Review
Xanthi Antoniou, Mattia Falconi, Daniele Di Marino, Tiziana BorselloJNK3 as a Therapeutic Target for Neurodegenerative Diseases
Abstract: c-Jun N-terminal kinases (JNKs), and in particular JNK3, the neuronal specific isoform, have been recognized as important enzymes in the pathology of diverse neurological disorders. Indeed, several efforts have been made to design drugs that inhibit JNK signaling. The success that characterized the new generation of cell permeable peptides raised the hopes of those in the field of neurodegeneration. However, in order to design new and more efficient therapeutic approaches, careful re-examination of current knowledge is required. Scaffold proteins are key endogenous regulators of JNK signaling: they can modulate spatial and temporal activation of the JNK signaling and can thus provide the basis for the design of more specific inhibitors. This review focuses on delineating the role of scaffold proteins on the regulation of JNK signaling in neurons. Furthermore the possibility to design a new JNK3 cell permeable peptide inhibitor by targeting the β-arrestin-JNK3 interaction is discussed.

Pages 643-656Review
Alan M. PalmerThe Role of the Blood Brain Barrier in Neurodegenerative Disorders and Their TreatmentAbstract: Neurodegenerative disorders represent a major medical challenge that is set to increase substantially in the decades ahead with the massive increase in the number of people in the world aged 65 or more. Neuroprotective therapeutics have the potential to play a key role in helping manage this growing global burden of long-term neurological care. However, neuropharmaceutical research is associated with significant challenges including: 1) the complexity of the brain (the cause of the majority of neurodegenerative disorders remains unknown); 2) the liability of central nervous system (CNS) drugs to cause CNS side effects (which limits their use); and 3) the requirement of neuropharmaceuticals to cross the blood-brain barrier (BBB). The BBB itself also plays a key role in most (if not all) neurodegenerative disorders since BBB dysfunction inevitably leads to inflammatory change including the movement of immune cells and immune mediators into the brain, which then contribute to the process of neurodegeneration. This review focuses on the role of the BBB in both neurodegenerative disorders and neuropharmaceutical research.

Pages 657-668Hypothesis
Jack C. de la Torre (Handling Associate Editor: Patrizia Mecocci)Three Postulates to Help Identify the Cause of Alzheimer’s Disease Abstract: Two centuries ago, the German bacteriologist Robert Koch proposed three postulates to support a causal relationship between a specific microbe and an infectious disease. Similarly, three postulates are formulated here to help evaluate hypothetical proposals attempting to explain the pathogenesis of Alzheimer’s disease (AD). The first postulate requires that the cause of AD precedes the cognitive decline and neurodegenerative pathology that characterize AD. This rule identifies a primary event from a neuropathological effect generated by the disease process. The second postulate stipulates that interventions aimed at the proposed causal event should prevent or reverse the cognitive and neurodegenerative pathology associated with AD prior to disease onset. This postulate emphasizes prevention or reversal of emerging neurocognitive pathology considerably before AD onset. If the first and second postulate requirements are met, the third postulate follows that interventions targeting the causal event should significantly lower the incidence of AD. For a causal hypothesis to be considered “likely” pathogenic to AD, support from all three postulates is a requisite. The pragmatic potential of the three postulates was applied to seven proposals using evidence-based meta-analysis mainly from randomized controlled trials. Proposals included the amyloid-β, cell cycle, cholinergic, inflammatory, oxidative stress, tau, and vascular hypotheses. Clinical evidence derived from each proposal formed the basis for an inferential conclusion based on the level of confidence provided by the trial data. The three postulates may challenge or help validate a proposed cause-effect relationship to AD and serve as a useful model for designing more intelligent therapeutic interventions aimed at preventing AD.

Pages 669-679Laura Mateos, Muhammad-Al-Mustafa Ismail, Francisco-Javier Gil-Bea, Valerio Leoni, Bengt Winblad, Ingemar Björkhem, Angel Cedazo-Mínguez (Handling Associate Editor: Miia Kivipelto)Upregulation of brain renin angiotensin system by 27-hydroxycholesterol in Alzheimer’s diseaseAbstract: In spite of the fact that cholesterol does not pass the blood-brain barrier, hypercholesterolemia has been linked to increase Alzheimer’s disease (AD) risk. Hypertension is another risk factor and angiotensin converting enzyme (ACE) activity is known to be increased in AD. Furthermore, a lower incidence of AD has been reported in patients taking anti-hypertensive drugs. Here we show that the levels of angiotensinogen (AGT) and ACE are increased in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment and AD. Moreover, we show ACE activity in the CSF to be positively correlated with both plasma and CSF levels of 27-hydroxycholesterol (27-OH), an oxysterol known to pass through the blood-brain barrier and taken up from the circulation by the brain. In addition, treatment of rat primary neurons, astrocytes, and human neuroblastoma cells with 27-OH resulted in increased production of AGT. Our results demonstrate that upregulation of renin-angiotensin system (RAS) in AD brains occurs not only at the enzymatic level (ACE) but also at the substrate level (AGT). The possibility that 27-OH is part of a mechanism linking hypercholesterolemia with increased brain RAS activity and increased AD risk is discussed.

Pages 681-691Ian T. Marsden, Laurie S. Minamide, and James R. BamburgAmyloid-β-Induced Amyloid-β Secretion: A Possible Feed-Forward Mechanism in Alzheimer’s Disease Abstract: Amyloid-β (Aβ) peptides, 36-43 amino acids in length, are produced from β- and γ-secretase cleavage of the amyloid-β protein precursor (AβPP), and are one of the causative agents of Alzheimer’s disease (AD). Here we show that an ELISA can detect total rodent Aβ without interference from physiological concentrations of human Aβ. In cultured dissociated rat cortical neurons and rat and mouse hippocampal organotypic slices, we apply the assay to measure the production of Aβ in response to treatment with hydrogen peroxide, a known stimulator of Aβ secretion, or human Aβ dimer/trimer (Aβd/t), fractionated from the culture medium of 7PA2 cells. Peroxide increases Aβ secretion by about 2 fold, similar to results from previous reports that used a different assay. Of greater significance is that physiologically relevant concentrations (~250 pM) of human Aβd/t increase rodent Aβ secretion from cultured rat cortical neurons by >3 fold over 4 days. Surprisingly, neither treatment with peroxide nor human Aβd/t leads to accumulation of intracellular Aβ. Human Aβd/t increased >2 fold the Aβ secreted by organotypic hippocampal slices from tau knock-out mice whether or not they expressed a human tau transgene, suggesting tau plays no role in enhanced Aβ secretion. Together, these results support an Aβ-mediated feed-forward mechanism in AD progression.

Pages 693-704Olena Lykhmus, Lyudmyla Koval, Maryna Skok, Marios Zouridakis, Paraskevi Zisimopoulou, Socrates Tzartos, Victor Tsetlin, Sylvie Granon, Jean-Pierre Changeux, Sergiy Komisarenko, Isabelle Cloëz-TayaraniAntibodies against Extracellular Domains of alpha4 and alpha7 Subunits Alter the Levels of Nicotinic Receptors in the Mouse Brain and Affect Memory: Possible Relevance to Alzheimer’s Pathology Abstract: Nicotinic acetylcholine receptors (nAChRs) of α4β2 and α7 subtypes expressed in the brain neurons are involved in regulating memory and cognition. Their level is decreased upon several neurodegenerative disorders including Alzheimer’s disease (AD), although the reasons for such a decrease are not completely understood. To test whether the nAChR-specific antibodies can affect the brain nAChRs and influence the behavior, we either immunized mice with recombinant extracellular domains of α4 and α7, subunits α4 (1-209) and α7 (1-208), or injected them with α7 (1-208)-specific antibodies. A decrease of α4β2- and α7-nAChRs accompanied with an increase of α4β4-nAChRs in brain membranes of immunized mice was observed. Both α4(1-209)- and α7(1-208)-specific antibodies were detected in the brain membrane lysates of immunized mice. Antibody injection resulted in brain nAChR decrease only if mice were co-injected intraperitoneally with bacterial lipopolysaccharide. Brain sections of immunized mice were analyzed for the binding of [125I]-α-bungarotoxin and [125I]-epibatidine.A decrease in α-bungarotoxin binding in striatum (nucleus accumbens and caudate putamen) accompanied with an increase of epibatidine binding in the forebrain and caudate putamen was observed in mice immunized with either α4 or α7 nAChR domains compared to those immunized with BSA. Mice immunized with α7(1-208) demonstrated significantly worse episodic memory measured in a novel object recognition task compared to non-immunized animals but did not differ from the controls in locomotor or anxiety-related tests. These results suggest that nAChR-specific antibodies are able to penetrate the brain upon inflammation with resulting decreases of brain nAChRs and worsening episodic memory.

Pages 705-719Apostolia Fragkouli, Athina K. Tzinia, Ioannis Charalampopoulos, Achille Gravanis, Effie C. Tsilibary (Handling Associate Editor: Elliott Mufson)Matrix Metalloproteinase-9 Participates in NGF-Induced α-Secretase Cleavage of Amyloid-β Protein Precursor in PC12 CellsAbstract: Amyloid-β protein precursor (AβPP) is a ubiquitously expressed glycoprotein, which under physiological conditions can be cleaved following two alternative routes; the non-amyloidogenic and the amyloidogenic pathway. Shift of AβPP processing in favor of the amyloidogenic pathway is a key event in the pathogenesis of Alzheimer’s disease (AD). Among the factors that regulate AβPP processing, nerve growth factor (NGF) appears to play an important role; abnormal NGF signaling has been implicated in the onset of AD. In the present study, we used PC12 cells to study the effects of NGF on AβPP processing and provide evidence that NGF, through binding to its high affinity receptor, TrkA moderately down-regulates the expression of the β-secretase β-site AβPP cleaving enzyme-1 and, most importantly, upregulates the expression of two enzymes with α-secretase activity, a disintegrin and metalloprotease-17 and to a greater extent matrix metalloproteinase-9 (MMP9) in a phosphoinositide kinase-3 dependent manner. Finally, we demonstrate that MMP9 actively participates in NGF-induced α-secretase cleavage of AβPP, thus it contributes to the shift of AβPP processing towards the non-amyloidogenic pathway precluding the formation of neurotoxic Aβ peptides.

Pages 721-738Ivan Arisi, Mara D’Onofrio, Rossella Brandi, Armando Felsani, Simona Capsoni, Guido Drovandi, Giovanni Felici, Emanuel Weitschek, Paola Bertolazzi, Antonino Cattaneo (Handling Associate Editor:Roberta Ghidoni)Gene Expression Biomarkers in the Brain of a Mouse Model for Alzheimer’s Disease: Mining of Microarray Data by Logic Classification and Feature Selection Abstract: The identification of early and stage-specific biomarkers for Alzheimer’s disease (AD) is critical, as the development of disease-modification therapies may depend on the discovery and validation of such markers. The identification of early reliable biomarkers depends on the development of new diagnostic algorithms to computationally exploit the information in large biological datasets. To identify potential biomarkers from mRNA expression profile data, we used the Logic Mining method for the unbiased analysis of a large microarray expression dataset from the anti-NGF AD11 transgenic mouse model. The gene expression profile of AD11 brain regions was investigated at different neurodegeneration stages by whole genome microarrays. A new implementation of the Logic Mining method was applied both to early (1-3 months) and late stage (6-15 months) expression data, coupled to standard statistical methods. A small number of “fingerprinting” formulas was isolated, encompassing mRNAs whose expression levels were able to discriminate between diseased and control mice. We selected three differential “signature” genes specific for the early stage (Nudt19, Arl16, Aph1b), five common to both groups (Slc15a2, Agpat5, Sox2ot, 2210015, D19Rik, Wdfy1), and seven specific for late stage (D14Ertd449, Tia1, Txnl4, 1810014B01Rik, Snhg3, Actl6a, Rnf25). We suggest these genes as potential biomarkers for the early and late stage of AD-like neurodegeneration in this model and conclude that Logic Mining is a powerful and reliable approach for large scale expression data analysis. Its application to large expression datasets from brain or peripheral human samples may facilitate the discovery of early and stage-specific AD biomarkers.

Pages 739-749Xin Zhang, Xin Heng, Ting Li, Lixi Li, Dehua Yang, Xiaojie Zhang, Yunlan Du, Rachelle S. Doody, Weidong LeLong-Term Treatment with Lithium Alleviates Memory Deficits and Reduces Amyloid-β Production in an Aged Alzheimer’s Disease Transgenic Mouse ModelAbstract: The glycogen synthase kinase-3β (GSK3β) pathway plays a central role in Alzheimer’s disease (AD) and its deregulation accounts for many of the pathological hallmarks of AD. Lithium, which modulates GSK3β activity, has been shown to reduce amyloid production and tau phosphorylation in pre-pathological AD mouse models. In this study, we investigate the effects of chronic LiCl treatment in aged double transgenic mice (AβPPSwe/PS1A246E). We found that chronic lithium treatment decreased the γ-cleavage of amyloid-β protein precursor, further reduced amyloid-β production and senile plaque formation. Spatial learning and memory abilities also improved. Because autophagy may play an important role in the pathology of AD, we also assessed the degree of autophagy after lithium treatment, and found that the chronic lithium treatment attenuates the autophagy activation in this AD mouse model. Our results suggest that prolonged lithium treatment, even during the later stages of AD, could be an effective therapeutic.

Pages 751-758Minerva M. Carrasquillo*, Olivia Belbin*, Talisha A. Hunter, Li Ma, Gina D. Bisceglio, Fanggeng Zou, Julia E. Crook, V. Shane Pankratz, Sigrid B. Sando, Jan O. Aasly, Maria Barcikowska, Zbigniew K. Wszolek, Dennis W. Dickson, Neill R. Graff-Radford, Ronald C. Petersen, Kevin Morgan for the Alzheimer’s Research Trust Consortium (ART), Steven G. Younkin (Handling Associate Editor: Francesco Panza) *These authors contributed equally to this work.Replication of BIN1 association with Alzheimer’s disease and evaluation of genetic interactions Abstract: The most recent late-onset Alzheimer’s disease (LOAD) genome-wide association study revealed genome-wide significant association of two new loci: rs744373 near BIN1 (p=1.6x10-11) and rs597668 near EXOC3L2/BLOC1S3/MARK4 (p=6.5x10-9). We have genotyped these variants in a large (3,287 LOAD, 4,396 controls), independent dataset comprising eleven case-control series from the USA and Europe. We performed meta-analyses of the association of these variants with LOAD and also tested for association using logistic regression adjusted by age-at-diagnosis, gender, and APOE ε4 status. Meta-analysis results showed no evidence of series heterogeneity and logistic regression analysis successfully replicated the association of BIN1 (rs597668) with LOAD with an odds ratio (OR=1.17, p=1.1x10-4) comparable to that previously reported (OR=1.15). The variant near EXOC3L2 (rs597668) showed only suggestive association with LOAD (p=0.09) after correcting for the presence of the APOE ε4 allele. Addition of our follow-up data to the results previously reported increased the strength of evidence for association with BIN1 (11,825 LOAD, 32,570 controls, rs744373 Fisher combined p=3.8x10-20). We also tested for epistatic interaction between these variants and APOE ε4 as well as with the previously replicated LOAD GWAS genes (CLU: rs11136000, CR1; rs3818361, and PICALM: rs3851179). No significant interactions between these genes were detected. In summary, we provide additional evidence for the variant near BIN1 (rs744373) as a LOAD risk modifier, but our results indicate that the effect of EXOC3L2 independent of APOE ε4 should be studied further.

Pages 759-766Masaya Arai, Mitsunari Saito, Hirokatsu Takatsu, Koji Fukui, Shiro Urano (Handling Associate Editor: Alexander Boldyrev)Dysfunction of the Fusion of Pre-Synaptic Plasma Membranes and Synaptic Vesicles Caused by Oxidative Stress, and Its Prevention by Vitamin E Abstract: To define whether hyperoxia induces the dysfunction of membrane fusion between synaptic vesicles with pre-synaptic plasma membranes in the
nerve terminals, and whether vitamin E prevents this abnormal event, we investigated the influence of hyperoxia on the fusion ability of isolated synaptic vesicles and the inside-out type pre-synaptic plasma membrane vesicles from rat brain using the fluorescence tracing method . The membrane fusion ability of both membranes from rats subjected to hyperoxia was markedly decreased compared with the membranes from a normal rat. Rats subjected to hyperoxia in the form of oxidative stress showed significant increases in the levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes, and protein carbonyl moieties in both synaptic vesicles and pre-synaptic plasma membranes. When rats were supplemented with vitamin E, these abnormalities were inhibited even when rats were subjected to hyperoxia.

Pages 775-783Wenlu Yang, Ronald L.M. Lui, Jia-Hong Gao, Tony F. Chan, Shing-Tung Yau, Reisa A. Sperling, Xudong HuangIndependent Component Analysis-Based Classification of Alzheimer’s Disease MRI Data Abstract: There is an unmet medical need to identify neuroimaging biomarkers that allow us to accurately diagnose and monitor Alzheimer’s disease (AD) at its very early stages and to assess the response to AD-modifying therapies. To a certain extent, volumetric and functional magnetic resonance imaging (fMRI) studies can detect changes in structure, cerebral blood flow, and blood oxygenation that distinguish AD and mild cognitive impairment (MCI) subjects from healthy control (HC) subjects. However, it has been challenging to use fully automated MRI analytic methods to identify potential AD neuroimaging biomarkers. We have thus proposed a method based on independent component analysis (ICA) for studying potential AD-related MR image features that can be coupled with the use of support vector machine (SVM) for classifying scans into categories of AD, MCI, and HC subjects. The MRI data were selected from the Open Access Series of Imaging Studies (OASIS) and the Alzheimer’s Disease Neuroimaging Initiative databases. The experimental results showed that the ICA method coupled with SVM classifier can differentiate AD and MCI patients from HC subjects, although further methodological improvement in the analytic method and inclusion of additional variables may be required for optimal classification.

Pages 785-797Roberto Tiribuzi, Antonio Orlacchio, Lucia Crispoltoni, Mariangela Maiotti, Mauro Zampolini, Massimiliano De Angeliz, Patrizia Mecocci, Roberta Cecchetti, Giorgio Bernardi, Alessandro Datti, Sabata Martino, Aldo Orlacchio (Handling Associate Editor: Annamaria Confaloni)Lysosomal β-Galactosidase and β-Hexosaminidase Activities Correlate with Clinical Stages of Dementia Associated with Alzheimer’s Disease and Type 2 Diabetes Mellitus Abstract: Multiple epidemiological studies have shown that individuals affected by type-2 diabetes mellitus (T2DM) carry a 2-to-5-fold higher risk of developing Alzheimer's disease (AD) when compared to non-diabetic subjects. Thus, biochemical parameters that can be easily and routinely assessed for high-confidence evaluation of diabetic conditions leading to AD (AD-T2DM) are regarded as efficient tools aimed at early diagnosis and, in turn, timely AD treatment. In this regard, the activity of lysosomal glycohydrolases may of use, in light of the implication of these enzymes in early events that underlie AD pathology and an overt correlation, in diabetes, between altered metabolic homeostasis, abnormal glycohydrolase secretion in body fluids, and occurrence of diabetic complications. Based on marked up-regulation previously shown in a peripheral, cell-based model of AD, we selected β-Galactosidase, β-Hexosaminidase, and α-Mannosidase to discriminate T2DM from AD-T2DM subjects. A screen of 109, 114, and 116 patients with T2DM, AD and AD-T2DM, respectively, was performed by testing enzyme activities in both blood plasma and peripheral blood mononuclear cells. Compared to age-matched, healthy controls (n=122), β-Galactosidase and β-Hexosaminidase activities markedly diverged across the three groups, whereas virtually unchanged values were observed for α-Mannosidase. In particular, plasma β-Galactosidase and β-Hexosaminidase levels were higher in patients with AD-T2DM compared to those with T2DM, suggesting different mechanisms leading to enzyme secretion. Statistical analyses based on ROC curves showed that both β-Galactosidase and β-Hexosaminidase activities, either intracellular or plasma-secreted, may be used to discriminate AD patients from controls and AD-T2DM from T2DM patients.

Pages 799-816Claudia Balducci, Bisan Mehdawy, Lydia Mare, Alessandro Giuliani, Luca Lorenzini, Sandra Sivilia, Luciana Giardino, Laura Calzà, Annamaria Lanzillotta, Ilenia Sarnico, Marina Pizzi, Alessandro Usiello, Arturo R. Viscomi, Simone Ottonello, Gino Villetti, Bruno P. Imbimbo, Giuseppe Nisticò, Gianluigi Forloni, Robert Nisticò (Handling Associate Editor: Rudy Castellani)The γ-Secretase Modulator CHF5074 Restores Memory and Hippocampal Synaptic Plasticity in Plaque-Free Tg2576 MiceAbstract: Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimer’s disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈ 60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment.We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

Pages 817-835Valentina Echeverria, Ross Zeitlin, Sarah Burgess, Sagar Patel, Arghya Barman, Garima Thakur, Magorzota Mamcarz, Li Wang, David B. Sattelle, Daniel A Kirschner, Takashi Mori, Roger M. Leblanc, Rajeev Prabhakar, Gary W. Arendash Cotinine Reduces Amyloid-β Aggregation and Improves Memory in Alzheimer’s Disease MiceAbstract: Alzheimer’s disease (AD) affects millions of people world-wide and new effective and safe therapies are needed. Cotinine, the main metabolite of nicotine, has a long half-life and does not have cardiovascular or addictive side effects in humans. We studied the effect of cotinine on amyloid-β (Aβ) aggregation as well as addressed its impact on working and reference memories. Cotinine reduced Aβ deposition, improved working and reference memories, and inhibited Aβ oligomerization in the brains of transgenic (Tg) 6799 AD mice. In vitro studies confirmed the inhibitory effect of cotinine on Aβ1-42 aggregation. Cotinine stimulated Akt signaling, including the inhibition of glycogen synthase kinase 3β (GSK3β), which promotes neuronal survival and the synaptic plasticity processes underlying learning and memory in the hippocampus and cortex of wild type and Tg mice. Simulation of the cotinine–Aβ1-42 complex using molecular dynamics showed that cotinine may interact with key histidine residues of Aβ1-42, altering its structure and inhibiting its aggregation. The good safety profile in humans and its beneficial effects suggest that cotinine may be an excellent therapeutic candidate for the treatment of AD.